Reverse isoxazoles

ABSTRACT

The present invention relates to polycyclic compounds, processes for their production, their use as pharmaceuticals and to pharmaceutical compositions comprising them.

This application is a U.S. National Phase filing of InternationalApplication Ser. No. PCT/EP2007/000638 filed 25 Jan. 2007, and claimspriority to G.B. application Ser. No. 0601744.6 filed 27 Jan. 2006, thecontents of which are incorporated herein by reference in theirentirety.

The present invention relates to polycyclic compounds, processes fortheir production, their use as pharmaceuticals and to pharmaceuticalcompositions comprising them.

More particularly the present invention provides in a first aspect acompound of formula I

whereineither X is —N═ or ═CH—;

R₁ is substituted biphenylyl, 4-phenoxy-phenyl or4-(phenyl-C₁₋₄alkoxy)-phenyl wherein at least one of the phenyl groupsis monosubstituted, phenyl substituted by one or more substituents orsubstituted 5 or 6-membered heteroaryl;

wherein the substituents in each of the above are independent from eachother selected from C₁₋₈alkyl, haloC₁₋₈alkyl, C₁₋₈alkoxy,haloC₁₋₈alkoxy, C₁₋₈alkoxy-C₁₋₈alkoxy, C₁₋₈alkyl-C₁₋₈alkoxy,C₁₋₈alkyl-haloC₁₋₈alkoxy, haloC₁₋₈alkyl-C₁-alkoxy,haloC₁₋₈alkyl-haloC₁₋₁₈alkoxy, haloC₁₋₈alkoxy-C₁₋₈alkoxy,C₁₋₈alkoxy-1-haloC₁₋₈alkoxy, haloC₁₋₈alkoxy-haloC₁₋₈alkoxy,C₁₋₈alkoxy-C₁₋₈alkyl, haloC₁₋₈alkoxy-C₁₋₈alkyl,C₁₋₈alkoxy-haloC₁₋₈alkyl, haloC₁₋₈alkoxy-haloC₁₋₈alkyl, C₂₋₆alkenyloxy,C₂₋₆alkynyloxy, C₃₋₆cycloalkyl, C₃₋₆cycloalkyl-C₁₋₄alkyl,C₃₋₆cycloalkyl-C₁₋₄alkoxy, C₃₋₄cycloalkyl-oxy, phenyl-C₁₋₄alkoxy andheterocyclic-C₁₋₄alkoxy;

R₂ is C₁₋₄ alkyl optionally substituted by halogen, OH, NH₂, C₁₋₄alkoxyor C₁₋₄alkylcarbonyloxy; amino; OH; C₁₋₄alkoxy; NH—OH; carboxy;sulfamoyl; carbamoyl; or HN—CO—C₁₋₄alkyl; or

R₂ is R₃-R₄—COOH or R₃-R₄—CONH₂ wherein R₃ is SO₂—NH, SO₂—N(C₁₋₄alkyl),CO—NH, CO—N(C₁₋₄alkyl), CH₂—O, NH—CO, or N(C₁₋₄alkyl)CO; and R₄ isC₁₋₆alkylene optionally interrupted by O, S or C═CH₂ or optionallysubstituted phenylene or C₃₋₆cycloalkylene; and

Ring A may be phenyl or heteroaryl, each being optionally furthersubstituted by one or more substituents independently selected fromhalogen, C₁₋₄alkyl, haloC₁₋₄alkyl, C₁₋₄alkoxy, haloC₁₋₄alkoxy ornitrile; or

R₂ is hydrogen under the proviso that Ring A is 3-pyridyl;

or a physiologically hydrolysable derivative thereof, a salt, hydrateand/or solvate thereof.

Halogen may be fluorine, chlorine or bromine, preferably fluorine orchlorine. Alkyl or alkoxy as a group or present in a group may bestraight or branched. C₁₋₆alkylene may be straight or branched.

HaloC₁₋₈alkyl or haloC₁₋₈alkoxy as a group or a moiety present in agroup may be C₁₋₈alkyl or C₁₋₈alkoxy substituted by 1 to 5 halogen, e.g.CF₃ or CF₃—CH₂—O—. C₁₋₈alkyl-haloC₁₋₈alkoxy may be haloC₁₋₈alkoxyfurther substituted by C₁₋₄alkyl, e.g. in position 1. The same may applyto the other groups.

When R₁ is substituted biphenylyl, 4-phenoxy-phenyl or4-(phenyl-C₁₋₄alkoxy)-phenyl, either one and/or both phenyl moieties maybe substituted, e.g. mono- or di-substituted e.g. by halogen, C₁₋₄alkyl,C₁₋₄alkoxy, haloC₁₋₁₈alkyl, haloC₁₋₈alkoxy or nitrile. Preferably atleast one phenyl moiety of the biphenylyl, 4-phenoxy-phenyl or4-(phenyl-C₁₋₄alkoxy)-phenyl is monosubstituted, e.g. as indicatedabove. More preferably each phenyl moiety of the biphenylyl,4-phenoxy-phenyl or 4-(phenyl-C₁₋₄alkoxy)-phenyl is monosubstituted,e.g. as indicated above, e.g. by haloC₁₋₈alkyl, and optionally assubstitutent on the second phenyl moiety either halogen, C₁₋₄alkyl orC₁₋₄alkoxy or haloC₁₋₈alkyl.

When R₁ is substituted phenyl, it may be mono- or di-substituted. WhenR₁ is mono-substituted phenyl, said substituent may preferably behaloC₁₋₈alkyl, in particular being in meta position, especiallyrepresenting trifluoromethyl in meta position. When R₁ is disubstitutedphenyl, one substituent may preferably be haloC₁₋₈alkyl, in particulartrifluoromethyl, and the second substitutent may be C₁₋₈alkyl,haloC₁₋₈alkyl, C₁₋₈alkoxy, haloC₁₋₈alkoxy, C₁₋₈alkoxy-C₁₋₈alkoxy,C₁₋₈alkyl-C₁₋₈alkoxy, C₁₋₈alkyl-haloC₁₋₈alkoxy,haloC₁₋₈alkyl-C₁₋₈alkoxy, haloC₁₋₈alkyl-haloC₁₋₈alkoxy,haloC₁₋₈alkoxy-C₁₋₈alkoxy, C₁₋₈alkoxy-haloC₁₋₈alkoxy,haloC₁₋₈alkoxy-haloC₁₋₈alkoxy, C₁₋₈alkoxy-C₁₋₈alkyl,haloC₁₋₈alkoxy-C₁₋₈alkyl, C₁₋₈alkoxy-haloC₁₋₈alkyl,haloC₁₋₈alkoxy-haloC₁₋₈alkyl, C₂₋₆alkenyloxy, C₂₋₆alkynyloxy,C₃₋₆cycloalkyl, C₃₋₆cycloalkyl-C₁₋₄alkyl, C₃₋₆cycloalkyl-C₁₋₄alkoxy,C₃₋₆cycloalkyl-oxy, phenyl-C₁₋₄alkoxy or heterocyclic-C₁₋₄alkoxy,preferably C₁₋₈alkyl, haloC₁₋₈alkyl, C₁₋₈alkoxy, haloC₁₋₈alkoxy orC₃₋₆cycloalkyl, more preferably C₁₋₈alkyl, haloC₁₋₈alkyl, C₁₋₈alkoxy orC₃₋₆cycloalkyl, and in particular C₃₋₆cycloalkyl. When R₁ isdisubstituted phenyl the first substituent is preferably in meta and thesecond preferably in para position.

Examples of a 5 or 6-membered heteroaryl as R₁ include e.g. thienyl orfuryl. Preferred is thienyl. When R₁ is substituted heteroaryl, it ismono- or disubstituted, preferably disubstituted. The substituent(s) maybe e.g. haloC₁₋₈alkyl, e.g. CF₃, and/or phenyl optionally substituted byhalogen, C₁₋₄alkyl or C₁₋₄alkoxy.

When R₄ is optionally substituted phenylene or C₃₋₆cycloalkylene, it maybe 1,4-phenylene or C₃₋₆cycloalkylene, e.g. cyclohexylene, optionallysubstituted by halogen.

Ring A may optionally be further substituted, e.g. by halogen,C₁₋₄alkyl, haloC₁₋₄alkyl, C₁₋₄alkoxy, haloC₁₋₄alkoxy or nitrile,preferably by halogen, C₁₋₄alkyl, haloC₁₋₄alkyl, C₁₋₄alkoxy, orhaloC₁₋₄alkoxy, more preferably by halogen, C₁₋₄alkyl, haloC₁₋₄alkyl, orC₁₋₄alkoxy, and especially by halogen, C₁₋₄alkyl or C₁₋₄alkoxy. WhenRing A is heteroaryl, it may be a 6-membered aromatic ring comprisingone heteroatom selected from nitrogen, oxygen and sulfur, e.g. pyridyl,pyrimidinyl or pyrazinyl. When Ring A is heteroaryl it may be preferablypyrimidinyl or pyridyl, more preferably pyridyl, even more preferably3-pyridyl.

The following significances are preferred independently, collectively orin any combination or sub-combination:

-   -   i) R₁ is biphenylyl, 4-phenoxy-phenyl or        4-(phenyl-C₁₋₄alkoxy)-phenyl wherein at least one of the phenyl        groups bears a haloC₁₋₄alkyl, e.g. CF₃;    -   ii) R₁ is phenyl substituted by haloC₁₋₄alkyl, e.g. CF₃ and        optionally by a second substituent as indicated above:    -   iii) R₁ is thienyl substituted by haloC₁₋₄alkyl, e.g. CF₃, and        phenyl;    -   iv) R₂ is sulfamoyl;    -   v) R₂ is R₃-R₄—COOH; or R₃-R₄—CONH₂; more preferably        R₃-R₄—CONH₂;    -   vi) R₃ is SO₂—NH; SO₂—N(C₁₋₄alkyl); NH—CO; or N(C₁₋₄alkyl)CO;    -   vii) R₄ is C₁₋₆alkylene optionally interrupted by O;    -   viii) R₂ is NH₂;    -   ix) Ring A is unsubstituted phenyl (i.e. no further substituent        in addition to R₂).    -   x) Ring A is substituted or unsubstituted (i.e. no further        substituent in addition to R₂) pyridyl.

The compounds of formula I may exist in free form or in salt form, e.g.addition salts with e.g. organic or inorganic acids, for example,hydrochloric acid or acetic acid, or salts obtainable when R₂ is orcomprises COOH, with a base, e.g. alkali salts such as sodium orpotassium, or substituted or unsubstituted ammonium salts.

It will be appreciated that the compounds of formula I may exist in theform of optical isomers, racemates or diastereoisomers. For example, R₄may comprise an asymmetric carbon atom when R₄ is branched alkylene. Itis to be understood that the present invention embraces all enantiomersand conformers and their mixtures. Similar considerations apply inrelation to starting materials exhibiting asymmetric carbon atoms asmentioned above.

By a physiologically hydrolysable derivative of a compound of formula Iis meant a compound which is hydrolysable under physiological conditionsto yield a compound of formula I and a by-product which is itselfphysiologically acceptable, e.g. an ester which is hydrolyzed to yield acompound of formula I and a non-toxic alcohol at the desired dosagelevels.

The present invention also includes a process for the production of acompound of formula I, which process comprises

-   -   a) for the production of a compound of formula I wherein X is        —N═ and R₂ is as defined above,    -   reacting a compound of formula II

-   -   wherein ring A and R₂ are as defined above, or a functional        derivative thereof, e.g. an activated ester, acyl chloride or        anhydride    -   with a compound of formula III

-   -   -   wherein R₁ is as defined above or a functional derivative            thereof; or

    -   b) for the production of a compound of formula I wherein X is CH        and R₂ is NH₂ reacting a compound of formula VI

-   -   wherein R₁ is as defined above, with a compound of formula VII

-   -   wherein Ring A is as defined above and R′₂ is NH₂; or    -   c) converting a compound of formula I into another compound of        formula I, and recovering the resulting compound of formula I in        free form or in form of a salt, and, where required converting        the compound of formula I obtained in free form into the desired        salt form or vice versa.

The process steps a) to c) may be performed according to methods knownin the art, or as disclosed below in the Examples.

Examples of conversion of a compound of formula I into another compoundof formula I may include e.g.

-   i) For the production of a compound of formula I wherein R₁ is    substituted biphenylyl, 4-phenoxy-phenyl or    4-(phenyl-C₁₋₄alkoxy)-phenyl wherein at least one of the phenyl    groups is monosubstituted, converting a compound of formula I    wherein R₁ is other than substituted biphenylyl, 4-phenoxy-phenyl or    4-(phenyl-C₁₋₄alkoxy)-phenyl wherein at least one of the phenyl    groups is monosubstituted, into a compound of formula I wherein R₁    is substituted biphenylyl, 4-phenoxy-phenyl or    4-(phenyl-C₁₋₄alkoxy)-phenyl wherein at least one of the phenyl    groups is monosubstituted.-   ii) For the production of a compound of formula I wherein R₂ is    R₃-R₄—COOH, hydrolyzing a compound of formula I wherein the COOH    present in R₂ is in form of a physiologically hydrolysable ester,    e.g. a methyl ester.-   iii) For the production of a compound of formula I wherein X is —N═    and Y is O and R₂ is R₃-R₄—COOH wherein R₃ is NH—CO or    N(C₁₋₄alkyl)CO and R₄ is as defined above, reacting a compound of    formula I

-   -   wherein R₁, X, R′₂ and ring A are as defined above, with an        acylating agent.

-   iv) For the production of a compound of formula I wherein R₂ is    R₃-R₄—CONH₂, reacting a compound of formula I wherein R₂ is    R₃-R₄—COOH with an amidating agent.

The compound of formula III used as starting material in process step a)may be obtained by reacting a compound of formula VIII

wherein R₁ is as defined above, with hydroxylamine.

The compound of formula VI used as starting material in process step b)may be produced by reacting a compound of formula IXR₁—COH  IXwherein R₁ is as defined above, with hydroxylamine.

A compound of formula I wherein X is —N═ and R₂ is NH₂ may also beproduced by reacting a compound of formula III with a compound offormula II′

wherein Ring A is as defined above, or a functional derivative thereof,e.g. an activated ester, acyl chloride or anhydride. The nitro grouppresent in the resulting compound may then be reduced, e.g. by catalytichydrogenation.

Insofar as the production of the starting materials is not particularlydescribed, the compounds are either known or may be prepared analogouslyto methods known in the art or as disclosed hereinafter.

The following Examples are illustrative of the invention.

EXAMPLE 14-[3-(2-trifluoromethyl-biphenyl-4-yl)-isoxazol-5-yl]-phenylamine

a) 2-Trifluoromethyl-biphenyl-4-carbaldehyde oxime

-   Step a) To a solution of 4-cyano-2-trifluoroaniline (1 eq) in    benzene there is added under inert atmosphere n-pentylnitrite (1 eq)    at 50° C. After one hour refluxing a second equivalent of    n-pentylnitrite is added. After additional two hours of refluxing    the reaction mixture is cooled to room temperature and concentrated    under reduced pressure. The dark residue is purified on silica gel    using c-hexane→-hexane/ethyl acetate 9/1 as mobile phase (pale    orange oil).-   Step b) The compound of step a) (1 eq) is dissolved in formic acid    (75%) and Ra—Ni (4 eq) is added. After 3 hours at 80° C. the    reaction mixture is filtered through Hyflo Super Cel® and the    catalyst/Hyflo is washed 2 times with ethanol (with    caution→flammable). The resulting solution is concentrated (yield    61%; mixture of ester & acid) and is used for step c) without any    further purification.-   Step c) The compound of step b) and hydroxylamine hydrochloride    (1.25 eq) are dissolved in ethanol and K₂CO₃ (1.1 eq) is added.    After 18 hours at room temperature the reaction mixture is    concentrated and after addition of water extracted with ethyl    acetate. Purification is achieved, after drying over Na₂SO₄ over    silica gel using c-hexane/ethyl acetate 9/1 as mobile phase,    yielding 2-trifluoromethyl-biphenyl-4-carbaldehyde oxime.

ESI-MS (ESI⁺): 266 (M+1H)⁺

b) 4-[3-(2-Trifluoromethyl-biphenyl-4-yl)-isoxazol-5-yl]-phenylamine

To a solution of 2-trifluoromethyl-biphenyl-4-carbaldehyde oxime (1 eq)in CH₂Cl₂ a 10% aqueous solution of NaOCl is added at 0° C. Thereafter asolution of 4-ethynylaniline (1.1 eq) is added and then the reactionmixture is stirred at room temperature for 16 hours. The reactionmixture is diluted with CH₂Cl₂ and 3 times extracted with water. Thecombined organic layers are dried over Na₂SO₄, filtered andconcentrated. Purification is achieved, after drying over Na₂SO₄, oversilica gel using c-hexane/ethyl acetate 9/1→7/3 as mobile phase.

ESI-MS (ESI⁺): 381 (M+1H)⁺

EXAMPLE 2N-{4-[3-(2-trifluoromethyl-biphenyl-5-yl)-isoxazol-3-yl]-phenyl}-succinamicacid

The compound (1 eq) of Example 1 is dissolved in CH₂Cl₂ and4-methylmorpholine (2 eq) and succinic anhydride (2 eq) are added. After16 hours at room temperature pure title product is obtained afterfiltration (white solid).

ESI-MS (ESI⁺): 481 (M+1H)⁺

EXAMPLE 3N-{4-[3-(2-trifluoromethyl-biphenyl-4-yl)-isoxazol-5-yl]-phenyl}-succinamide

The compound of Example 2 is dissolved in DMF and subsequentlyN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC.HCl;1.5 eq), hydroxybenzotriazole (HOBt; 1.3 eq), NH₄OH 25% in water (1.2eq) and diisopropylethylamine (1.5 eq) are added. After 16 hours at roomtemperature the reaction mixture is concentrated and purified on silicagel (CH₂Cl₂/methanol 95/5→CH₂Cl₂/methanol/acetic acid_(50%) 90/10/0.125as mobile phase) resulting in pure title compound.

ESI-MS (ESI⁺): 480 (M+1H)⁺

EXAMPLE 43-{4-[3-(2-Trifluoromethyl-biphenyl-4-yl)-[1,2,4]oxadiazol-5-yl]-benzene-sulfonylamino}-propionicacid

a) N-Hydroxy-2-trifluoromethyl-biphenyl-4-carboxamidine

-   Step a) To a solution of 4-cyano-2-trifluoroaniline (1 eq) in    benzene there is added under inert atmosphere n-pentylnitrite (1 eq)    at 50° C. After one hour refluxing a second equivalent of    n-pentylnitrite is added. After additional two hours of refluxing    the reaction mixture is cooled to room temperature and concentrated    under reduced pressure. The dark residue is purified on silica gel    using c-hexane→c-hexane/ethyl acetate 9/1 as mobile phase (pale    orange oil).-   Step b) The compound of step a) is dissolved in THF and    hydroxylamine (50% in water; 10 eq) is added dropwise at −10° C.    After 16 hours at room temperature the reaction mixture is diluted    with water and extracted with ethyl acetate. After drying of the    organic phase with Na₂SO₄ purification is achieved on silica gel    with c-hexane/ethyl acetate 75/25 as mobile phase.

b) 4-(2-Methoxycarbonyl-ethylsulfamoyl)-benzoic acid

It is obtained by reacting 4-chlorosulfonyl-benzoic acid (1 eq) withH-βAla-OMe×HCl in CH₂Cl₂ using diisopropylethylamine (2 eq) as base.After 30 minutes at room temperature the reaction mixture is extractedwith water and the organic layer is dried over Na₂SO₄. Removal of thesolvent gives the title compound.

c)3-{4-[3-(2-Trifluoromethyl-biphenyl-4-yl)-[1,2,4]oxadiazol-5-yl]-benzenesulfonylamino}-propionicacid methyl ester

4-(2-Methoxycarbonyl-ethylsulfamoyl)-benzoic acid (1 eq) is dissolved inDMF and EDC.HCl (1.3 eq) and HOBt (1.1 eq) are added. After 30 minutesat room temperature N-hydroxy-2-trifluoromethyl-biphenyl-4-carboxamidine(1 eq) is added and the reaction mixture is kept at 90° C. for 16 hours.After removal of the solvent the residue is dissolved in ethyl acetateand extracted with saturated NaHCO₃ solution. Title compound is obtainedafter drying of the organic phase over Na₂SO₄ on silica gel usingc-hexane/ethyl acetate/CH₂Cl₂ 8/2/1 as mobile phase.

ESI-MS (ESI⁻): 530 (M−1H)⁻

d)3-{4-[3-(2-Trifluoromethyl-biphenyl-4-yl)-[1,2,4]oxadiazol-5-yl]-benzene-sulfonylamino}-propionicacid

LiOH (2 eq) is dissolved in methanol/water (1/1) and the ester (1 eq) isadded. After 4 hours at 50° C. methanol is removed under reducedpressure, the pH is adjusted to ˜3 with 1N HCl and the reaction mixtureis 3 times extracted with ethyl acetate. The combined organic layers aredried over Na₂SO₄, filtered, concentrated and the title compound isobtained as a white powder after removal of the solvent.

ESI-MS (ESI⁻): 516 (M−1H)⁻

EXAMPLE 54-[3-(2-Trifluoromethyl-biphenyl-4-yl)-[1,2,4]oxadiazol-5-yl]-phenylamine

a)5-(4-Nitro-phenyl)-3-(2-trifluoromethyl-biphenyl-4-yl)-[1,2,4]oxadiazole

It is obtained as a white powder using 4-nitrobenzoic acid instead of4-(2-methoxycarbonyl-ethylsulfamoyl)-benzoic acid in the procedure ofExample 1—step c).

ESI-MS (ESI⁺): 412 (M+1H)⁺

b) The compound of step a) is dissolved in methanol/ethyl acetate 1/1and hydrogenated at room temperature under normal pressure for 16 hourswith Pd/C_(10%) as catalyst. After filtration through Hyflo Super Cel®the reaction mixture is concentrated and purified on silica gel(CH₂Cl₂→CH₂Cl₂/methanol 95/5 as mobile phase), yielding the titlecompound.

ESI-MS (ESI⁺): 382 (M+1H)⁺

EXAMPLE 62,6-Dimethoxy-3-[3-(2-trifluoromethyl-biphenyl-4-yl)-[1,2,4]oxadiazol-5-yl]-pyridine

2,6-Dimethoxy-nicotinic acid (1 eq) is dissolved in dioxane and EDC.HCl(1.3 eq) and HOBt (1.1 eq) are added. After 30 minutes at roomtemperature N-hydroxy-2-trifluoromethyl-biphenyl-4-carboxamidine (1 eq)is added and the reaction mixture is kept at 900 degrees Celsius for 16hours. After removal of the solvent the residue is dissolved in ethylacetate and extracted with saturated NaHCO₃ solution. Title compound isobtained after drying of the organic phase over Na₂SO₄ on silica gelusing c-hexane/ethyl acetate→c-hexane/ethyl acetate 100→70/30 as mobilephase.

ESI-MS (ESI⁻): 426 (M−1H)⁻

By following the procedure as described in the foregoing Examples andusing the appropriate starting materials, the compounds of formula I

wherein X, R₁, R₂ and Ring A are as defined in Table 1 below, areobtained.

TABLE 1 Ex R₁ X R₂ Ring A ESI + MS: 7 2-CF₃-4- CH HN—CO—CH₂—C(CH₃)₂—COOHphenyl 509 biphenylyl (M + 1H)⁺ 8 2-CF₃-4- CH HN—CO—CH₂—C(CH₃)₂—CONH₂phenyl 508 biphenylyl (M + 1H)⁺ 9 3-CF₃-4- CH NH₂ phenyl 387 cyclohexyl-(M + 1H)⁺ phenyl 10 3-CF₃-4- CH HN—CO—CH₂—CH₂—COOH phenyl 487cyclohexyl- (M + 1H)⁺ phenyl 11 3-CF₃-4- CH HN—CO—CH₂—CH₂—CONH₂ phenyl486 cyclohexyl- (M + 1H)⁺ phenyl 12 2-CF₃-4- N SO₂—NH—CH₂—CH₂—CONH₂phenyl 515 biphenylyl (M − 1H)⁻ 13 2-CF₃-4- N SO₂—NH₂ phenyl 444biphenylyl (M − 1H)⁻ 14 2-CF₃-4- N OH phenyl 383 biphenylyl (M − 1H)⁻ 152-CF₃-4- N OH 3-CF₃- 451 biphenylyl phenyl (M − 1H)⁻ 16 2-CF₃-4- NHN—CO—CH₂—CH₂—COOH phenyl 480 biphenylyl (M − 1H)⁻ 17 2-CF₃-4- NHN—CO—CH₂—C(CH₃)₂—COOH phenyl 508 biphenylyl (M − 1H)⁻ BHT482 182-CF₃-4- N HN—CO—CH₂—CH₂—CONH₂ phenyl 479 biphenylyl (M − 1H)⁻ 192-CF₃-4- N HN—CO—CH₂—C(CH₃)₂—CONH₂ phenyl 507 biphenylyl (M − 1H)⁻ 203-CF₃-4- N NH₂ phenyl 388 cyclohexyl- (M + 1H)⁺ phenyl 21 3-CF₃-4- NHN—CO—CH₂—CH₂—COOH phenyl 488 cyclohexyl- (M + 1H)⁺ phenyl 22 3-CF₃-4- NHN—CO—CH₂—CH₂—CONH₂ phenyl 487 cyclohexyl- (M + 1H)⁺ phenyl 23 2-CF₃-4-N H 3-pyridyl 368 biphenylyl (M + 1H)⁺ 24 2-CF₃-4- N NH₂ 3-pyridyl 383biphenylyl (M + 1H)⁺ 25 2-CF₃-4- N OH 3-pyridyl 384 biphenylyl (M + 1H)⁺26 2-CF₃-4- N NH—OH 3-pyridyl 399 biphenylyl (M + 1H)⁺ 27 2-CF₃-4- NHN—CO—CH₂—CH₂—COOH 3-pyridyl 483 biphenylyl (M + 1H)⁺ 28 2-CF₃-4- N COOH3-pyridyl 412 biphenylyl (M + 1H)⁺ 29 3-CF₃-4- N COOH 3-pyridyl 418cyclohexyl- (M + 1H)⁺ phenyl 30 2-CF₃-4- N COOH 2-pyridyl 412 biphenylyl(M + 1H)⁺ 31 2-CF₃-4- N CH₂—NH₂ phenyl 396 biphenylyl (M + 1H)⁺

The compounds of formula I in free form or in pharmaceuticallyacceptable salt form, exhibit valuable pharmacological properties, e.g.as S1P1 receptor agonists, e.g. as indicated in in vitro and in vivotests and are therefore indicated for therapy.

A. In vitro

The compounds of formula I have binding affinity to individual human S1Preceptors as determined in following assays:

A. In vitro: GPCR Activation Assay Measuring GTP [γ-³⁵S] Binding toMembranes Prepared from CHO Cells Expressing Human EDG Receptors

S1P₁ (EDG-1) GTP [γ-³⁵S] binding assay: Homogenized membranes areprepared from CHO cell clones stably expressing a human EDG-1 N-terminalc-myc tag. Cells are grown in suspension in two 850 cm² roller bottlesfor three or fours days before harvesting. The cells are centrifugeddown, washed once with cold PBS, and resuspended in ≦20 ml of Buffer A(20 mM HEPES, pH 7.4, 10 mM EDTA, EDTA-free complete protease inhibitorcocktail [1 tablet/25 ml]). The cell suspension is homogenized on ice,using a Polytron homogenizer at 30000 rpm at three intervals of 15seconds each. The homogenate is first centrifuged at 2000 rpm on atabletop low speed centrifuge for 10 minutes. The supernatant, afterpassing through a cell strainer, is then re-centrifuged at 50,000×g for25 minutes at 4° C. The pellet is resuspended into buffer B (15%glycerol, 20 mM HEPES, pH 7.4, 0.1 mM EDTA, EDTA-free complete proteaseinhibitor cocktail [1 tablet/10 ml]). Protein concentration of thepreparation is determined using the BCA Protein Assay kit (Pierce) usingBSA as standard. The membranes are aliquoted and kept frozen at −80° C.

Solutions of test compounds ranging from 10 mM to 0.01 nM are preparedin DMSO. S1P is diluted in 4% BSA solution as positive controls. Thedesired amount of membrane preparation is diluted with ice-cold assaybuffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl₂, 0.1% Fattyacid-free BSA, 5 μM GDP) and vortexed well. 2 μl or less of compound isdistributed into each well of a round-bottom 96-well polystyrene assayplate, followed by addition of 100 μl of diluted membranes (3-10μg/well) and kept on ice until the addition of hot GTPγS. [³⁵S]-GTPγS isdiluted 1:1000 (v/v) with cold assay buffer and 100 μl is added intoeach well. The reaction is carried out at room temperature for 90minutes before the membranes are harvested onto Perkin-Elmer Unifilter®GF/B-96 filter plate using a Packard Filtermate Harvester. After severalwashes with wash buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl₂₎,and a rinse with 95% ethanol, the filter is dried in a 37° C. oven for30 minutes. MicroScint-20 is added and the plate sealed forscintillation counting on TopCount. EC₅₀ values are obtained by fittingthe GTP [γ-³⁵S] binding curves (raw data) with the dose responsecurve-fitting tool of GraphPad Prism. Six or twelve differentconcentrations are used to generate a concentration response curve(using three data points per concentration).

S1P 3, -4, -5 and -6 GTP [γ-³⁵S] binding assays are carried out in acomparable manner to the S1P1 GTP [γ-³⁵S] binding assay using membranesfrom CHO cells stably expressing c-terminal c-myc tagged or untaggedreceptors. For each membrane preparation, titration experiments arefirst run with S1P control to determine the optimal amount of membranesto be added per assay well.

Compounds of formula I are tested according to the above assay and areobserved to exhibit selectivity for the S1P1 receptor. For example,Compounds of Examples 4, 13 and 21 have an EC₅₀<1 μM in the above assayand are e.g. at least 20 fold selective for S1P1 compared to S1P3, ande.g. at least 20 fold selective for S1P1 compared to S1P5.

B. In vitro: FLIPR Calcium Flux Assay

Compounds of the invention are tested for agonist activity on S1P1,S1P3, S1P5, and S1P6 with a FLIPR calcium flux assay. Briefly, CHO cellsexpressing an S1P receptor are maintained in F-12K medium (ATCC),containing 5% FBS, with 500 μg/ml of G418. Prior to the assay, the cellsare plated in 384 black clear bottom plates at the density of 10,000cells/well/25 μl, in the medium of F-12K containing 1% FBS. The secondday, the cells are washed three times (25 μl/each) with washing buffer.About 25 μl of dye are added to each well and incubated for 1 hour at37° C. and 5% CO₂. The cells are then washed four times with washingbuffer (25 μl/each). The calcium flux is assayed after adding 25 μl ofSEW2871 (published by Rosen et al., used as reference) solution to eachwell of cells. The same assay is performed with cells expressing each ofthe different S1P receptors. Titration in the FLIPR calcium flux assayis recorded over a 3-minute interval, and quantitated as maximal peakheight percentage response relative to S1P-1 activation. The compoundsof the invention are active in this assay at a concentration of from10⁻¹² and 3.10⁻⁵ nM.

C. In vivo: Screening Assays for Measurement of Blood LymphocyteDepletion

Measurement of circulating lvmphocytes: Compounds to be tested aredissolved in DMSO/PEG200 and further diluted with deionized water. Rats(Lewis strain, female, 6-12 weeks old) are administered 1 mg/kg ofcompound to be tested in 4 ml/kg vehicle (max. 2% DMSO/max. 2%PEG200/water) via per os application. DMSO/PEG200/water and FTY720 (0.3mg/kg) are included as negative and positive controls, respectively.

Blood is collected from the sublingual vein 2, 6, 24 and 48 hours afteradministration under short isoflurane anesthesia. Whole blood samplesare subjected to hematology analysis. Peripheral lymphocyte counts aredetermined using an automated analyzer. Subpopulations of peripheralblood lymphocytes are stained by fluorochrome-conjugated specificantibodies and analyzed using a fluorescent activating cell sorter(Facscalibur). Two rats are used to assess the lymphocyte depletionactivity of each compound screened. The result is an ED₅₀, which isdefined as the effective dose required to display 50% of bloodlymphocyte depletion. Compounds of formula I are tested according to theabove assay and have an ED₅₀ of less than 10 mg/kg.

The compounds of formula I are, therefore, useful in the treatmentand/or prevention of diseases or disorders mediated by lymphocytesinteractions, e.g. in transplantation, such as acute or chronicrejection of cell, tissue or organ allo- or xenografts or delayed graftfunction, graft versus host disease, autoimmune diseases, e.g.rheumatoid arthritis, systemic lupus erythematosus, hashimoto'sthyroidis, multiple sclerosis, myasthenia gravis, diabetes type I or IIand the disorders associated therewith, vasculitis, pernicious anemia,Sjoegren syndrome, uveitis, psoriasis, Graves opthalmopathy, alopeciaareata and others, allergic diseases, e.g. allergic asthma, atopicdermatitis, allergic rhinitis/conjunctivitis, allergic contactdermatitis, inflammatory diseases optionally with underlying aberrantreactions, e.g. inflammatory bowel disease, Crohn's disease orulcerative colitis, intrinsic asthma, inflammatory lung injury,inflammatory liver injury, inflammatory glomerular injury,atherosclerosis, osteoarthritis, irritant contact dermatitis and furthereczematous dermatitises, seborrhoeic dermatitis, cutaneousmanifestations of immunologically-mediated disorders, inflammatory eyedisease, keratoconjunctivitis, myocarditis or hepatitis,ischemia/reperfusion injury, e.g. myocardial infarction, stroke, gutischemia, renal failure or hemorrhage shock, traumatic shock, cancer,e.g. breast cancer, T cell lymphomas or T cell leukemias, infectiousdiseases, e.g. toxic shock (e.g. superantigen induced), septic shock,adult respiratory distress syndrome or viral infections, e.g. AIDS,viral hepatitis, chronic bacterial infection, or senile dementia.Examples of cell, tissue or solid organ transplants include e.g.pancreatic islets, stem cells, bone marrow, corneal tissue, neuronaltissue, heart, lung, combined heart-lung, kidney, liver, bowel,pancreas, trachea or oesophagus. For the above uses the required dosagewill of course vary depending on the mode of administration, theparticular condition to be treated and the effect desired.

In general, satisfactory results are indicated to be obtainedsystemically at daily dosages of from about 0.03 to 5.0 mg/kg per bodyweight. An indicated daily dosage in the larger mammal, e.g. humans, isin the range from about 0.5 mg to about 500 mg, convenientlyadministered, for example, in divided doses up to four times a day or inretard form. Suitable unit dosage forms for oral administration comprisefrom ca. 0.1 to 50 mg active ingredient.

The compounds of formula I may be administered by any conventionalroute, in particular enterally, e.g. orally, e.g. in the form of tabletsor capsules, or parenterally, e.g. in the form of injectable solutionsor suspensions, topically, e.g. in the form of lotions, gels, ointmentsor creams, or in a nasal or a suppository form. Pharmaceuticalcompositions comprising a compound of formula I in free form or inpharmaceutically acceptable salt form in association with at least onepharmaceutical acceptable carrier or diluent may be manufactured inconventional manner by mixing with a pharmaceutically acceptable carrieror diluent.

The compounds of formula I may be administered in free form or inpharmaceutically acceptable salt form e.g. as indicated above. Suchsalts may be prepared in conventional manner and exhibit the same orderof activity as the free compounds.

In accordance with the foregoing the present invention further provides:

-   1.1 A method for preventing or treating disorders or diseases    mediated by lymphocytes, e.g. such as indicated above, in a subject    in need of such treatment, which method comprises administering to    said subject an effective amount of a compound of formula I or a    pharmaceutically acceptable salt thereof;-   1.2 A method for preventing or treating acute or chronic transplant    rejection or T-cell mediated inflammatory or autoimmune diseases,    e.g. as indicated above, in a subject in need of such treatment,    which method comprises administering to said subject an effective    amount of a compound of formula I or a pharmaceutically acceptable    salt thereof;-   2. A compound of formula I, in free form or in a pharmaceutically    acceptable salt form for use as a pharmaceutical, e.g. in any of the    methods as indicated under 1.1 or 1.2 above.-   3. A pharmaceutical composition, e.g. for use in any of the methods    as in 1.1 or 1.2 above comprising a compound of formula I in free    form or pharmaceutically acceptable salt form in association with a    pharmaceutically acceptable diluent or carrier therefor.-   4. A compound of formula I or a pharmaceutically acceptable salt    thereof for use in the preparation of a pharmaceutical composition    for use in any of the method as in 1.1 or 1.2 above.

The compounds of formula I may be administered as the sole activeingredient or in conjunction with, e.g. as an adjuvant to, other drugse.g. immunosuppressive or immunomodulating agents or otheranti-inflammatory agents, e.g. for the treatment or prevention of allo-or xenograft acute or chronic rejection or inflammatory or autoimmunedisorders, or a chemotherapeutic agent, e.g a malignant cellanti-proliferative agent. For example, the compounds of formula I may beused in combination with a calcineurin inhibitor, e.g. cyclosporin A orFK 506; a mTOR inhibitor, e.g. rapamycin,40-O-(2-hydroxyethyl)-rapamycin, CCI779, ABT578, AP23573, AP23464,AP23675, AP23841, TAFA-93, biolimus-7 or biolimus-9; an ascomycin havingimmunosuppressive properties, e.g. ABT-281, ASM981, etc.;corticosteroids; cyclophosphamide; azathioprene; methotrexate;leflunomide; mizoribine; mycophenolic acid or salt; mycophenolatemofetil; 15-deoxyspergualine or an immunosuppressive homologue, analogueor derivative thereof; a PKC inhibitor, e.g. as disclosed in WO 02/38561or WO 03/82859, e.g. the compound of Example 56 or 70; a JAK3 kinaseinhibitor, e.g. N-benzyl-3,4-dihydroxy-benzylidene-cyanoacetamideα-cyano-(3,4-dihydroxy)-]N-benzylcinnamamide (Tyrphostin AG 490),prodigiosin 25-C (PNU156804),[4-(4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline] (WHI-P131),[4-(3′-bromo-4′-hydroxylphenyl)amino-6,7-dimethoxyquinazoline](WHI-P154),[4-(3′,5′-dibromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline]WHI-P97, KRX-211,3-{(3R,4R)-4-methyl-3-[methyl-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-amino]-piperidin-1-yl}-oxo-propionitrile,in free form or in a pharmaceutically acceptable salt form, e.g.mono-citrate (also called CP-690,550), or a compound as disclosed in WO04/052359 or WO 05/066156; immunosuppressive monoclonal antibodies,e.g., monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3,CD4, CD7, CD8, CD25, CD28, CD40, CD45, CD52, CD58, CD80, CD86 or theirligands; other immunomodulatory compounds, e.g. a recombinant bindingmolecule having at least a portion of the extracellular domain of CTLA4or a mutant thereof, e.g. an at least extracellular portion of CTLA4 ora mutant thereof joined to a non-CTLA4 protein sequence, e.g. CTLA4lg(for ex. designated ATCC 68629) or a mutant thereof, e.g. LEA29Y;adhesion molecule inhibitors, e.g. LFA-1 antagonists, ICAM-1 or -3antagonists, VCAM-4 antagonists or VLA-4 antagonists; or achemotherapeutic agent, e.g. paclitaxel, gemcitabine, cisplatinum,doxorubicin or 5-fluorouracil; or an anti-infectious agent.

Where the compounds of formula I are administered in conjunction withother immunosuppressive/immunomodulatory, anti-inflammatory.chemotherapeutic or anti-infectious therapy, dosages of theco-administered immunosuppressant, immunomodulatory, anti-inflammatory,chemotherapeutic or anti-infectious compound will of course varydepending on the type of co-drug employed, e.g. whether it is a steroidor a calcineurin inhibitor, on the specific drug employed, on thecondition being treated and so forth. In accordance with the foregoingthe present invention provides in a yet further aspect:

-   5. A method as defined above comprising co-administration, e.g.    concomitantly or in sequence, of a therapeutically effective    non-toxic amount of a compound of formula I and at least a second    drug substance, e.g. an immunosuppressant, immunomodulatory,    anti-inflammatory or chemotherapeutic drug, e.g. as indicated above.-   6. A pharmaceutical combination, e.g. a kit, comprising a) a first    agent which is a compound of formula I as disclosed herein, in free    form or in pharmaceutically acceptable salt form, and b) at least    one co-agent, e.g. an immunosuppressant, immunomodulatory,    anti-inflammatory, chemotherapeutic or anti-infectious agent. The    kit may comprise instructions for its administration.

The terms “co-administration” or “combined administration” or the likeas utilized herein are meant to encompass administration of the selectedtherapeutic agents to a single patient, and are intended to includetreatment regimens in which the agents are not necessarily administeredby the same route of administration or at the same time.

The term “pharmaceutical combination” as used herein means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound of formula I and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g. a compound of formula I and a co-agent, are bothadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the 2compounds in the body of the patient. The latter also applies tococktail therapy, e.g. the administration of 3 or more activeingredients.

1. A compound of formula I:

wherein X is —N═ or ═CH—; R₁ is biphenylyl, 4-phenoxyphenyl or4-(phenyl-C₁-C₄-alkoxy)-phenyl, where at least one of the phenyl groupsbears a halo-C₁₋₄-alkyl; or is phenyl substituted by C₃₋₆-cycloalkyl andhalo-C₁₋₈-alkyl in the meta and para positions or vice versa; R₂ isC₁₋₄-alkyl optionally substituted by halogen, OH, NH₂, C₁₋₄-alkoxy orC₁₋₄-alkylcarbonyloxy; or is amino; OH; C₁₋₄-alkoxy; NH—OH; carboxy;sulfamoyl; carbamoyl; or HN—CO—C₁₋₄-alkyl; or R₂ is R₃-R₄—COOH orR₃-R₄—CONH₂, where R₃ is SO₂—NH, SO₂—N(C₁₋₄-alkyl), CO—NH,CO—N(C₁₋₄-alkyl), CH₂—O, NH—CO, or N(C₁₋₄-alkyl)CO, and R₄ isC₁₋₆-alkylene optionally interrupted by O; and Ring A is phenyloptionally further substituted by halogen, C₁₋₄-alkyl, halo-C₁₋₄-alkyl,C₁₋₄-alkoxy, halo-C₁₋₄-alkoxy or nitrile, or a salt thereof.
 2. Thecompound according to claim 1, wherein R₁ is biphenylyl, where at leastone of the phenyl groups bears a halo-C₁₋₄-alkyl; or is phenylsubstituted by halo-C₁₋₈-alkyl and C₃₋₆-cycloalkyl in the para and metapositions or vice versa, or a salt thereof.
 3. The compound according toclaim 1, wherein R₁ is biphenylyl, where at least one of the phenylgroups bears a halo-C₁₋₄-alkyl; or is phenyl substituted byC₃₋₆-cycloalkyl and halo-C₁₋₈-alkyl in the meta position and the paraposition or vice versa; and R₂ is R₃-R₄—COOH or R₃-R₄—CONH₂, where R₃ isSO₂—NH, SO₂—N(C₁₋₄-alkyl), CO—NH, CO—N(C₁₋₄-alkyl), CH₂—O, NH—CO, orN(C₁₋₄-alkyl)CO, and R₄ is C₁₋₆-alkylene optionally interrupted by O; ora salt thereof.
 4. A process for the preparation of a compound offormula I,

which process comprises (i) for the production of a compound of formulaI, wherein X is —N═ and R₂ is C₁₋₄-alkyl optionally substituted byhalogen, OH, NH₂, C₁₋₄-alkoxy or C₁₋₄-alkylcarbonyloxy; or is amino; OH;C₁₋₄-alkoxy; NH—OH; carboxy; sulfamoyl; carbamoyl; or HN—CO—C₁₋₄-alkyl;or is R₃-R₄—COOH or R₃-R₄—CONH₂, where R₃ is SO₂—NH, SO₂—N(C₁₋₄-alkyl),CO—NH, CO—N(C₁₋₄-alkyl), CH₂—O, NH—CO, or N(C₁₋₄-alkyl)CO, and R₄ isC₁₋₆-alkylene optionally interrupted by O, reacting a compound offormula II

wherein ring A is phenyl optionally further substituted by halogen,C₁₋₄-alkyl, halo-C₁₋₄-alkyl, C₁₋₄-alkoxy, halo-C₁₋₄-alkoxy or nitrile,and R₂ is as defined above, or a functional derivative thereof, with acompound of formula III

wherein R₁ is biphenylyl, 4-phenoxyphenyl or4-(phenyl-C₁-C₄-alkoxy)-phenyl, where at least one of the phenyl groupsbears a halo-C₁₋₄-alkyl; or is phenyl substituted by C₃₋₆-cycloalkyl andhalo-C₁₋₈-alkyl in the meta and para positions or vice versa, or afunctional derivative thereof; or (ii) for the production of a compoundof formula I, wherein X is CH and R₂ is NH₂, reacting a compound offormula VI

wherein R₁ is as defined above, with a compound of formula VII

wherein Ring A is as defined above and R₂′ is NH₂; and recovering eitherresulting compound of formula I in free form or in salt form, and, whererequired, converting the compound of formula I obtained in free forminto the desired salt form or vice versa.
 5. A pharmaceuticalcomposition comprising a compound according to claim 1 in free form orpharmaceutically-acceptable salt form in association with apharmaceutically-acceptable diluent or carrier therefor.
 6. Apharmaceutical combination, comprising a) a first agent which is acompound according to claim 1, in free form or inpharmaceutically-acceptable salt form, and b) at least one co-agent thatis an immunosuppressant, immunomodulatory, anti-inflammatory,chemotherapeutic or anti-infectious agent.
 7. The compound according toclaim 1 that is3-{4-[3-(2-trifluoromethyl-biphenyl-4-yl)-[1,2,4]oxadiazol-5-yl]-benzenesulfonylamino}-propionicacid, or a salt thereof.